The long term goal of the proposed research is to purify the enzymes which phosphorylate phosphatidylinositol (PI) and characterize their structure, mechanism of action and regulation. Recent results from several laboratories have implicated these enzymes as being important in mediation of hormone responses. The inositol-1,4,5-trisphosphate (IP3) which regulates free Ca2+ levels in the cytoplasm is derived from hydrolysis of phosphotidylinositol-4,5-bisphosphate (PIP2). In addition, since much of the cellular diacylglycerol (DG) derives from breakdown of PI, and since free Ca2+ levels appear to affect this breakdown, it seems likely that PIP2 breakdown is both directly and indirectly affecting the diacylglycerol level in cells. Thus, PIP2 is a critical intermediate in the pathway for production of two important second messengers. These second messengers have been proposed as mediators of a variety of hormone actions including proliferative responses to certain growth factors. We have proposed that an increase in the rate of phosphorylation of PI to phosphatidylinositol-4-phosphate (PIP) and to phosphatidylinositol-4,5-bisphosphate (PIP2) could enhance responses to hormones by providing additional substrate for production of the second messengers. We now wish to purify these enzymes to homogeneity and characterize their structure, substrate specificity, and mechanism of regulation.